Method and apparatus for the assay of viable microorganisms



Dec.

D. STEFANYE ETAL METHOD AND APPARATUS FOR THE ASSAY OF VIABLE MICROORGANISMS Filed Sept. 18, 1956 zzvmvrozes Dav/d .Sfefanye Jule N. Dews By W ATTORNE United States Patent METHOD AND APPARATUS FOR THE ASSAY OF VIABLE MICROORGANISMS David Stefanye and Jule N. Dews, Frederick, Md. Application September 18, 1956, Serial No. 610,695 7 Claims. 01. 19s 103.s

(Granted under Title 35, U. S. Code (1952), sec. 266) The invention described herein may be manufactured and used by or for the Government of the United States of America for governmental purposes Without the payment to us of any royalty thereon.

This invention relates to an apparatus for diluting an aerosol to enable a count to be made of the viable organisms therein. More specifically, the invention relates to a mechanism for preparing and diluting an aerosol by a known amount and of depositing the remaining or a proportion of the remaining organisms on a nutrient media for culturing and counting.

The established method for determining the number of viable organisms in a liquid isto introduce a measured amount of liquid into a large amount of water or other diluent and shake the resulting mixture. A sample is then withdrawn, rediluted and reshaken until a final degree of dilution is obtained such that a given volume of liquid may be plated out on a suitable media in a Petri dish and incubated. The resulting colonies are then counted and the original concentration determined from the dilution ratio. This method of determining the presence and number of viable organisms in a liquid has some disadvantages. It is quite slow, requiring much mixing, and the repeated sampling and redilution may be dangerous when toxic organisms are handled.

The present invention avoids these difii'culties by making an aerosol of a given quantity of liquid and rapidly diluting the same before any appreciable settling takes place. This procedure eliminates the mixing and shaking problem inherent in dilution in the liquid state. Moreover, the aerosol is continuously inside of the ap paratus during the entire determination so that no contamination can take place and the plating out on Petri dishes is automatically taken care of. Moreover, by utilizing a suitable atomizer or nebulizer, aerosol particle size approaches the dimensions of a single microorganism, thus causing chains and clusters of cells to separate, whereupon a more exact count is realized.

In Figure 1, beginning at the top, the five sections of the apparatus are shown in descending order at 2, 4, 6, 8 and 10. Atomizer inlet 12 in section 2 provides for the admission of the aerosol into the upper chamber. Stopcocks 26, 28, 30, 32 and 34 serve to connect the respective sections with line 35 for admitting a sterile gas to the system. In a similar manner, the respective compartments 2, 4, 6 and 8 are fitted with outlet connections 36, 38, 40 and 42, which are equipped with stopcocks 37, 39, 41 and 43 respectively. Corresponding inlet connections 52, 54, 56 and 58, together with stopcocks 53, 55, 57 and 59, are fitted to chambers 4, 6, 8 and 10. These inlet and outlet connections are joined to sub-chambers 44, 46, 48 and 50, the outlet from one chamber and inlet to the next lower chamber being connected to the same sub-chamber. By means of these several connections it is possible for an aerosol to be passed from one compartment to another via the subchambers and connections therebetween. The sub-cham- Patented 'Dec; 23 1 958 bers are vented into line 62 by means of stopcocks 45, 47, 49 and 51.

The respective chambers and sub-chambers may be of any reasonable size, but for convenience in calcula tions the following sizes were used:

By choosing these sizes,

the following volumetric relationships obtain:

Vol. 2+vol. 44:1,000ml. and vol. 44=a 10 dilution.

Vol. 4+vol. 44+vol. 46:1,000 ml. and vol. 46:21 10 dilution.

Vol. 6+vol. 46+vol. 48:1,000 ml. and vol. 48=a 10 dilution.

Vol. 8+vol. 48+vol. 50:1,000 ml. and vol. 50=a 10 dilution.

The apparatus may be constructed of glass, metal, ceramics or plastics, the only requirements being that it shall be airtight, capable of sustaining asubstantialvacuum and must be capable of being sterilized by steam,

heat, chemicals or gases. In addition, the walls should be smooth and not toxic to the organisms tested. In the preferred form the apparatus is made ofv glass having ground joints at 9 and 11.

The transverse shape of the present apparatus is trifoliate, although a circular or other cross section could also be used. The section shown was chosen since it permits the close fitting of three Petri-dishes as shown in Fig. 2. With this design, almost all of the aerosol settling out will fall on one or another of the dishes.

The two lower sections 8 and 10 are fitted with trans* verse sealed joints 9 and 11, thereby permitting the sections to be opened for: the placing or removing of the Petri dishes.

In operation, the apparatus is assembled and sterilized with steam, with all stopcocks open. The apparatus is then cooled to about 37 C. and the Petri dishes with their nutrient media are inserted in chambers 8 and 10. The purpose of having plates in both of the lower chambers is to obtain an adjacent count differing by a power of 10. Stopcocks 26, 28, 30, 32 and 34 are closed and the entire apparatus is evacuated through line 62 after which all stopcocks are closed. A measured amount of liquid containing viable organisms to be counted is now introduced into chamber 2 through the spray nozzle 12 as a finely divided aerosol. Sterile air or inert gas is admitted through stopcock 26 to bring chamber 2 to substantially atmospheric pressure. A sample of the aerosol is now taken into sub-chamber 44 by opening stopcock 37. Subchamber 44, being under a vacuum, will fill promptly from chamber 2, the latter being at atmospheric pressure. This latter sample now contains ,4 of the organisms originally present in chamber 2. Stopcocks 26 and 37 are closed and 53 opened, thereby transferring most of the sample in sub-chamber 44 to chamber 4. The total volume of the selected sample of aerosol is now volume 4+volume 44. Stopcock 28 is opened to bring about equilibrium with sterile air or gas as before. Stopcock 39 is then opened to transfer part of the sample to sub chamber 46. The total volume occupied by the selected aerosol sample is now vol. 4+vol. 44+vol. 46,

time, by means of the exhaust system.

This total volume has already been defined as 1,000 ml. and since vol. 46 is ml., there is again a dilution of 100 to 1 or a total of 10,000 to 1 (10 between vol. 46 and the initial aerosol volume.

Stopcocks 28 and 39 are now closed and 55 opened, thereby largely transferring the aerosol into chamber 6.

. Stopcock 30 is opened for pressure equilibrium as before and the sample partially transferred in succession to subchamber 48, then chamber 8, sub-chamber 50, and chamber 10.. The stabilization of pressures through stopcocks 26, 28, 30, 32 and 34 is not essential to correct results, but it assists the movement of aerosol from chamber to chamber by maintaining the required pressure differential. The manipulation of stopcocks should be as indicated. Stopcocks 26, 28, 30, 32 and 34 are closed in order as indicated to prevent line 35 from being contaminated from the content of the upper aerosol chambers. In each transfer of aerosol there is a specific dilution of 100 to 1 and in the last stage between chamber 8 and sub-chamber 50 there is a dilution of 10 to 1. This gives a multi-stage dilution of 10 10 10 and 10 or a total of 10' to 1.

This dilution is sufliciently high so that when the organisms reach chamber 10 they are so sparse that upon settling out on Petri dishes 60 and incubated, the resulting colonies can be counted. This count, multiplied by the dilution ratio, gives the organism count in the original liquid sample with a high degree of accuracy.

The use of settling out in both chambers 8 and 10 in a 10 to 1 ratio provides a check against the accuracy of the findings of the lower chamber. In practice it is not feasible to wait for the settling out of every viable particle in chamber 10. On the contrary, it is adequate to permit the greater number of particles to settle and then to remove the particles remaining after a specified In practice, a settling time of one-half hour results in an almost complete count of all viable particles. Since settling time varies for difierent size particles it is desirable to standardize the apparatus for different particle sizes. Thus, where particle size range of a given aerosol is known, this standardization permits of some economy of time in running the determination.

In operating the mechanism, the entire dilution procedure can be carried out in a matter of minutes and before any appreciable settling out takes place. After the multi-stage dilutioh has been carried out it will be noted that the several chambers are full of aerosol at the various concentrations obtained at that particular stage. Thus, the settling out that is allowed to take place in stage 8, in nowise affects the subsequent dilution into stage 10. In order to preclude extensive settling in stages 2, 4 and 6, the aerosol contents of these stages may be exhausted through 62 as soon as the aerosol dilution has been carried beyond that particular stage. This reduces somewhat the cleaning necessary in the various stages.

We claim:

1. A dilution apparatus comprising a series of sealed main volumetric stages, intermediate smaller substages connected in series with said main stages and having valved inlet and outlet connections between stages, each substage having additional valved outlet connections into a common conduit to permit evacuation of the apparatus.

2. A method of diluting and culturing an aerosol of viable organisms which comprises enclosing a volume of the aerosol, withdrawing a fractional decimal volume of the original volume, enlarging said smaller volume, withdrawing a further fractional decimal volume and repeating this process in subsequent stages until the aerosol is sufficiently dilute, allowing the diluted aerosol to settle out on a nutrient media, culturing and counting the resulting colonies and multiplying this value by the decimal dilution factor to secure the number of organisms in the original volume.

3. Apparatus in accordance with claim 1, wherein an additional vent is fitted to each main stage to permit direct admission of gas to said stage.

4. An apparatus in accordance with claim 1, wherein the last main stage is fitted with Petri dishes to enable the culturing of any organisms settling out thereon.

' 5. An apparatus in accordance with claim 4, wherein the apparatus is made of glass in a trifoliate section.

6. An apparatus in accordance with claim 1, wherein the first stage is fitted with an atomizer for introducing an aerosol into the stage.

7. An apparatus in accordance with claim 1, wherein the volumetric ratios of the various stages and substages are such that the ratio of the volume of the first substage to the sum of the volumes of the first stage and substage is a positive power of 10 and the ratio of the volume of subsequent substages to the sum of the volumes of the substage in question plus the previous stage and substage is a positive power of 10.

References Cited in the file of this patent UNITED STATES PATENTS 1,043,361 Romer Nov. 5, 1912 

1. A DILUTION APPARATUS COMPRISING A SERIES OF SEALED MAIN VOLUMETRIC STAGES, INTERMEDIATE SMALLER SUBSTAGES CONNECTED IN SERIES WITH SAID MAIN STAGES AND HAVING VALVED INLET AND OUTLET CONNECTIONS BETWEEN STAGES, EACH 